Aquaculture mediates global transmission of a viral pathogen to wild salmon.

Global expansion of aquaculture and agriculture facilitates disease emergence and catalyzes transmission to sympatric wildlife populations. The health of wild salmon stocks critically concerns Indigenous peoples, commercial and recreational fishers, and the general public. Despite potential impact of viral pathogens such as Piscine orthoreovirus-1 (PRV-1) on endangered wild salmon populations, their epidemiology in wild fish populations remains obscure, as does the role of aquaculture in global and local spread. Our phylogeographic analyses of PRV-1 suggest that development of Atlantic salmon aquaculture facilitated spread from Europe to the North and South East Pacific. Phylogenetic analysis and reverse transcription polymerase chain reaction surveillance further illuminate the circumstances of emergence of PRV-1 in the North East Pacific and provide strong evidence for Atlantic salmon aquaculture as a source of infection in wild Pacific salmon. PRV-1 is now an important infectious agent in critically endangered wild Pacific salmon populations, fueled by aquacultural transmission.

Discovery and surveillance of viruses from salmon in British Columbia using viral immune-response biomarkers, metatranscriptomics, and high-throughput RT-PCR.

The emergence of infectious agents poses a continual economic and environmental challenge to aquaculture production, yet the diversity, abundance, and epidemiology of aquatic viruses are poorly characterised. In this study, we applied salmon host transcriptional biomarkers to identify and select fish in a viral disease state, but only those that were negative for known viruses based on RT-PCR screening. These fish were selected for metatranscriptomic sequencing to discover potential viral pathogens of dead and dying farmed Atlantic (Salmo salar) and Chinook (Oncorhynchus tshawytscha) salmon in British Columbia (BC). We found that the application of the biomarker panel increased the probability of discovering viruses in aquaculture populations. We discovered two viruses that have not previously been characterised in Atlantic salmon farms in BC (Atlantic salmon calicivirus and Cutthroat trout virus-2), as well as partially sequenced three putative novel viruses. To determine the epidemiology of the newly discovered or emerging viruses, we conducted high-throughput reverse transcription polymerase chain reaction (RT-PCR) and screened over 9,000 farmed and wild salmon sampled over one decade. Atlantic salmon calicivirus and Cutthroat trout virus-2 were in more than half of the farmed Atlantic salmon we tested. Importantly we detected some of the viruses we first discovered in farmed Atlantic salmon in Chinook salmon, suggesting a broad host range. Finally, we applied in situ hybridisation to determine infection and found differing cell tropism for each virus tested. Our study demonstrates that continual discovery and surveillance of emerging viruses in these ecologically important salmon will be vital for management of both aquaculture and wild resources in the future.

Descriptive multi-agent epidemiology via molecular screening on Atlantic salmon farms in the northeast Pacific Ocean.

Rapid expansion of salmon aquaculture has resulted in high-density populations that host diverse infectious agents, for which surveillance and monitoring are critical to disease management. Screening can reveal infection diversity from which disease arises, differential patterns of infection in live and dead fish that are difficult to collect in wild populations, and potential risks associated with agent transmission between wild and farmed hosts. We report results from a multi-year infectious-agent screening program of farmed salmon in British Columbia, Canada, using quantitative PCR to assess presence and load of 58 infective agents (viruses, bacteria, and eukaryotes) in 2931 Atlantic salmon (Salmo salar). Our analysis reveals temporal trends, agent correlations within hosts, and agent-associated mortality signatures. Multiple agents, most notably Tenacibaculum maritimum, were elevated in dead and dying salmon. We also report detections of agents only recently shown to infect farmed salmon in BC (Atlantic salmon calicivirus, Cutthroat trout virus-2), detection in freshwater hatcheries of two marine agents (Kudoa thyrsites and Tenacibaculum maritimum), and detection in the ocean of a freshwater agent (Flavobacterium psychrophilum). Our results provide information for farm managers, regulators, and conservationists, and enable further work to explore patterns of multi-agent infection and farm/wild transmission risk.

Environmental DNA from multiple pathogens is elevated near active Atlantic salmon farms.

The spread of infection from reservoir host populations is a key mechanism for disease emergence and extinction risk and is a management concern for salmon aquaculture and fisheries. Using a quantitative environmental DNA methodology, we assessed pathogen environmental DNA in relation to salmon farms in coastal British Columbia, Canada, by testing for 39 species of salmon pathogens (viral, bacterial, and eukaryotic) in 134 marine environmental samples at 58 salmon farm sites (both active and inactive) over 3 years. Environmental DNA from 22 pathogen species was detected 496 times and species varied in their occurrence among years and sites, likely reflecting variation in environmental factors, other native host species, and strength of association with domesticated Atlantic salmon. Overall, we found that the probability of detecting pathogen environmental DNA (eDNA) was 2.72 (95% CI: 1.48, 5.02) times higher at active versus inactive salmon farm sites and 1.76 (95% CI: 1.28, 2.42) times higher per standard deviation increase in domesticated Atlantic salmon eDNA concentration at a site. If the distribution of pathogen eDNA accurately reflects the distribution of viable pathogens, our findings suggest that salmon farms serve as a potential reservoir for a number of infectious agents; thereby elevating the risk of exposure for wild salmon and other fish species that share the marine environment.

Endangered wild salmon infected by newly discovered viruses.

The collapse of iconic, keystone populations of sockeye (Oncorhynchus nerka) and Chinook (Oncorhynchus tshawytscha) salmon in the Northeast Pacific is of great concern. It is thought that infectious disease may contribute to declines, but little is known about viruses endemic to Pacific salmon. Metatranscriptomic sequencing and surveillance of dead and moribund cultured Chinook salmon revealed a novel arenavirus, reovirus and nidovirus. Sequencing revealed two different arenavirus variants which each infect wild Chinook and sockeye salmon. In situ hybridisation localised arenavirus mostly to blood cells. Population surveys of >6000 wild juvenile Chinook and sockeye salmon showed divergent distributions of viruses, implying different epidemiological processes. The discovery in dead and dying farmed salmon of previously unrecognised viruses that are also widely distributed in wild salmon, emphasizes the potential role that viral disease may play in the population dynamics of wild fish stocks, and the threat that these viruses may pose to aquaculture.

Distribution and Phylogeny of Erythrocytic Necrosis Virus (ENV) in Salmon Suggests Marine Origin.

Viral erythrocytic necrosis (VEN) affects over 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. However, the distribution and strain variation of its viral causative agent, erythrocytic necrosis virus (ENV), has not been well characterized within Pacific salmon. Here, metatranscriptomic sequencing of Chinook salmon revealed that ENV infecting salmon was closely related to ENV from Pacific herring, with inferred amino-acid sequences from Chinook salmon being 99% identical to those reported for herring. Sequence analysis also revealed 89 protein-encoding sequences attributed to ENV, greatly expanding the amount of genetic information available for this virus. High-throughput PCR of over 19,000 fish showed that ENV is widely distributed in the NE Pacific Ocean and was detected in 12 of 16 tested species, including in 27% of herring, 38% of anchovy, 17% of pollock, and 13% of sand lance. Despite frequent detection in marine fish, ENV prevalence was significantly lower in fish from freshwater (0.03%), as assessed with a generalized linear mixed effects model (p = 5.5 × 10-8). Thus, marine fish are likely a reservoir for the virus. High genetic similarity between ENV obtained from salmon and herring also suggests that transmission between these hosts is likely.

Detection and Assessment of the Distribution of Infectious Agents in Juvenile Fraser River Sockeye Salmon, Canada, in 2012 and 2013.

Infectious diseases may contribute to declines in Fraser River Sockeye salmon (Oncorhynchus nerka) stocks, but a clear knowledge gap exists around which infectious agents and diseases are important. This study was conducted to: (1) determine the presence and prevalence of 46 infectious agents in juvenile Fraser River Sockeye salmon, and (2) evaluate spatial patterns in prevalence and burden over initial seaward migration, contrasting patterns between 2 years of average and poor productivity. In total, 2,006 out-migrating Sockeye salmon were collected from four regions along their migration trajectory in British Columbia, in 2012 and 2013. High-throughput microfluidics quantitative PCR was employed for simultaneous quantitation of 46 different infectious agents. Twenty-six agents were detected at least once, including nine with prevalence >5%. Candidatus Brachiomonas cysticola, Myxobolus arcticus, and Pacific salmon parvovirus were the most prevalent agents. Infectious agent diversity and burden increased consistently upon smolts entry into the ocean, but they did not substantially change afterwards. Notably, both freshwater- and saltwater-transmitted agents were more prevalent in 2013 than in 2012, leading to an overall higher infection burden in the first two sampling regions. A reduction in the prevalence of two agents, erythrocytic necrosis virus and Paraneuclospora theridion, was observed between regions 2 and 3, which was speculated to be associated with mortality during the 1st month at sea. The most prevalent infectious agents were all naturally occurring. In a small number of samples (0.9%), seven agents were only detected around and after salmon farming regions, including four important pathogens: piscine orthoreovirus, Piscirickettsia salmonis, Tenacibaculum maritimum, and Moritella viscosa. As the first synoptic survey of infectious agents in juvenile Sockeye salmon in British Columbia, this study provides the necessary baseline for further research on the most prevalent infectious agents and their potential pathogenicity, which may adversely affect the productivity of valuable Sockeye salmon stocks. In addition, our findings are informative to the decision makers involved in conservation programs.

Infectious disease, shifting climates, and opportunistic predators: cumulative factors potentially impacting wild salmon declines.

Emerging diseases are impacting animals under high-density culture, yet few studies assess their importance to wild populations. Microparasites selected for enhanced virulence in culture settings should be less successful maintaining infectivity in wild populations, as once the host dies, there are limited opportunities to infect new individuals. Instead, moderately virulent microparasites persisting for long periods across multiple environments are of greatest concern. Evolved resistance to endemic microparasites may reduce susceptibilities, but as barriers to microparasite distributions are weakened, and environments become more stressful, unexposed populations may be impacted and pathogenicity enhanced. We provide an overview of the evolutionary and ecological impacts of infectious diseases in wild salmon and suggest ways in which modern technologies can elucidate the microparasites of greatest potential import. We present four case studies that resolve microparasite impacts on adult salmon migration success, impact of river warming on microparasite replication, and infection status on susceptibility to predation. Future health of wild salmon must be considered in a holistic context that includes the cumulative or synergistic impacts of multiple stressors. These approaches will identify populations at greatest risk, critically needed to manage and potentially ameliorate the shifts in current or future trajectories of wild populations.

Patterns of selection and allele diversity of class I and class II major histocompatibility loci across the species range of sockeye salmon (Oncorhynchus nerka).

The major histocompatibility complex (MHC), an important component of the vertebrate immune system, provides an important suite of genes to examine the role of genetic diversity at non-neutral loci for population persistence. We contrasted patterns of diversity at the two classical MHC loci in sockeye salmon (Oncorhynchus nerka), MHC class I (UBA) and MHC class II (DAB), and neutral microsatellite loci across 70 populations spanning the species range from Washington State to Japan. There was no correlation in allelic richness or heterozygosity between MHC loci or between MHC loci and microsatellites. The two unlinked MHC loci may be responding to different selective pressures; the distribution of FST values for the two loci was uncorrelated, and evidence for both balancing and directional selection on alleles and lineages of DAB and UBA was observed in populations throughout the species range but rarely on both loci within a population. These results suggest that fluctuating selection has resulted in the divergence of MHC loci in contemporary populations.

Rediscovery of the Yesso scallop pathogen Perkinsus qugwadi in Canada, and development of PCR tests.

Perkinsus qugwadi, a pathogenic protozoan parasite of Yesso scallops Patinopecten yessoensis, is found only in cultured populations in British Columbia, Canada. This pathogen was first identified in 1988 and caused significant mortalities at some locations during the early 1990s. Prevalence of infection decreased dramatically following 1995, and the disease was last reported in 1997, leading to speculation that the Yesso scallop stocks in Canada had developed resistance to the disease, or that P. qugwadi had disappeared. However, the present study revealed that infection with P. qugwadi and associated mortality is still occurring in scallops from at least one location in British Columbia. One of the PCR tests developed for P. qugwadi detected the parasite in a 105-fold dilution of DNA extracted from a heavily infected sample and detected 52% more positive scallops than histology; however, the assay also cross-reacted with P. honshuensis and P. olseni. The other PCR test was less sensitive and detected 34% more positives, but did not react to any of the other Perkinsus species tested, suggesting that these PCR tests are powerful tools for screening for the presence of P. qugwadi. Phylogenetic analysis of 1796 bp of SSU rRNA gene sequence clearly indicated that P. qugwadi is positioned basally to other Perkinsus species.

Sequence analysis of MHC class I α2 from sockeye salmon (Oncorhynchus nerka).

Most studies assessing adaptive MHC diversity in salmon populations have focused on the classical class II DAB or DAA loci, as these have been most amenable to single PCR amplifications due to their relatively low level of sequence divergence. Herein, we report the characterization of the classical class I UBA α2 locus based on collections taken throughout the species range of sockeye salmon (Oncorhynchus nerka). Through use of multiple lineage-specific primer sets, denaturing gradient gel electrophoresis and sequencing, we identified thirty-four alleles from three highly divergent lineages. Sequence identity between lineages ranged from 30.0% to 56.8% but was relatively high within lineages. Allelic identity within the antigen recognition site (ARS) was greater than for the longer sequence. Global positive selection on UBA was seen at the sequence level (dN:dS = 1.012) with four codons under positive selection and 12 codons under negative selection.